Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia.

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

[Suspicion of constitutional abnormality at diagnosis of childhood leukemia: Update of the leukemia committee of the French Society of Childhood Cancers]. / Suspicion d'anomalie constitutionnelle au diagnostic de leucémie chez l'enfant : mise au point du comité leucémies de la Société française des cancers de l'enfant.

The spectrum of childhood leukemia predisposition syndromes has grown significantly over last decades. These predisposition syndromes mainly involve CEBPA, ETV6, GATA2, IKZF1, PAX5, RUNX1, SAMD9/SAMD9L, TP53, RAS-MAPK pathway, DNA mismatch repair system genes, genes associated with Fanconi anemia, and trisomy 21. The clinico-biological features leading to the suspicion of a leukemia predisposition are highly heterogeneous and require varied exploration strategies. The study of the initial characteristics of childhood leukemias includes high-throughput sequencing techniques, which have increased the frequency of situations where a leukemia predisposing syndrome is suspected. Identification of a leukemia predisposition syndrome can have a major impact on the choice of chemotherapy, the indication for hematopoietic stem cell transplantation, and screening for associated malformations and pathologies. The diagnosis of a predisposition syndrome can also lead to the exploration of family members and genetic counseling. Diagnosis and management should be based on dedicated and multidisciplinary care networks.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

Cytogenetics in the management of hematologic neoplasms with germline predisposition: guidelines from the Groupe Francophone de Cytogénétique Hématologique (GFCH).

The number of predisposing genes is continuously growing with the widespread availability of DNA sequencing, increasing the prevalence of hematologic malignancies with germline predisposition. Cytogenetic analyses provide an effective approach for the recognition of these malignancies with germline predisposition, which is critical for proper diagnosis, optimal treatment and genetic counseling. Based on the World Health Organization and the international consensus classifications as well as the European LeukemiaNet recommendations, this review first presents an advanced classification of neoplasms with germline predisposition focused on the acquired cytogenetic alterations during leukemogenesis. The various genetic rescue mechanisms and the progression to transformation are then explained. The review also outlines the specific constitutional and somatic cytogenetic aberrations indicative of germline predisposition disorders in B-acute lymphoblastic leukemia (ALL), T-ALL, bone marrow failure syndrome and myeloid neoplasms. An emphasis is made on monosomy 7 in the predisposition field, its frequency and diagnosis impact as well as its various circumstances of occurrence. Lastly, we propose cytogenetic technical recommendations and guidelines for clinical reporting of these specific aberrations.

Hereditary predisposition to malignant myeloid hemopathies: Caution in use of saliva and guideline based on our experience.

Background: Predisposition to myeloid malignancies is a field at the border of hematology and genetics. Knowledge in this domain has so rapidly increased that WHO defined in 2016 the new "Myeloid Neoplasms with Germline Predisposition" category of tumors. High throughput sequencing is frequently performed in tumors either for diagnosis or prognosis, but this approach may identify potential germline variants that have to be confirmed on non-infiltrated tissues. Method: In this study, we systematically compared NGS data from genetic analysis performed on all sample types (bone marrow, blood, saliva, skin fibroblasts and hair follicles) in 29 patients, and 44 of their relatives (blood and saliva). Results: We showed that saliva was usable for relatives, but only for 24% (7/29) of our patients. Most of patients' saliva were either "non-contributive" (14/29 i.e., 48% because clearly or probably infiltrated) or "inconclusive" (8/29 corresponding to 28%). Conclusion: The recommendations for the use of saliva we present here focus on the importance of collecting saliva during remission when possible. Moreover, we propose hair follicles as an alternative to skin biopsy, that remains the gold standard especially in case of allogenic hematopoietic stem cells transplantation. Technological progresses have revolutionized the diagnosis of predisposition to solid or hematological malignancies, and it is very likely that new techniques will help to manage the familial predisposition in the future.

Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia.

Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.

Flow cytometry detection of CD138 expression continuum between monotypic B and plasma cells is associated with both high IgM peak levels and MYD88 mutation and contributes to diagnosis of Waldenström macroglobulinemia.

BACKGROUND: Differential diagnosis of Waldenström macroglobulinemia (WM) with other indolent B-cell malignancies is still a challenge. Here, we propose an original and simple analysis of routine flow cytometry (FCM) unraveling the characteristic ongoing plasma cell (PC) differentiation of WM tumor B-cells. METHODS: FCM analysis of both B-cells and PC was performed on a series of 77 patients with IgM peak. MYD88 and CXCR4 mutations were studied using an allele-specific PCR and by high throughput sequencing. RESULTS: Twenty seven (35%), 46 (58%) and 4 (5%) patients were classified as WM, IgM monoclonal gammopathy of undetermined significance (MGUS) or other B-NHL respectively. MYD88 mutation was found in 25/27 WM (93%) and in 29/46 MGUS (63%). Using FCM, monotypic B-cells were found in 27/27 WM (100%) and 34/46 MGUS (74%). Monotypic CD138pos/CD38pos PCs were detected in 23/27 WM (85%) and 25/46 MGUS (54%). Highlighting the ongoing PC differentiation of WM tumor B-cells by FCM, we evidenced a CD138 expression continuum between monotypic B-cells and PCs. This pattern remained absent in control samples and was significantly associated with higher IgM peaks (p = 6.10-5 ) and MYD88 mutations (p = 10-3 ) in both WM and MGUS cases. CONCLUSIONS: FCM exploration of both B-cells and PC led to identify a CD138 expression continuum as an objective marker of ongoing PC differentiation of WM tumor cells and was strongly associated with increased IgM peak levels and MYD88 mutations. This approach could contribute to place FCM at the forefront of WM diagnosis.

CD20 expression, TrkB activation and functional activity of diffuse large B cell lymphoma-derived small extracellular vesicles.

BACKGROUND: Small extracellular vesicles (sEVs) including exosomes, carrying the CD20, could be involved in immunotherapy resistance in diffuse large B cell lymphoma (DLBCL). We have reported endogenous brain-derived neurotrophic factor/TrkB (tropomyosin-related kinase B) survival axis in DLBCL. Here, we performed a comparative study of sEV production by germinal centre B cell (GCB) and activated B cell (ABC)-DLBCL cell lines, and analysed TrkB activation on this process. METHODS: GCB (SUDHL4 and SUDHL6) and ABC (OCI-LY3, OCI-LY10 and U2932) cell lines were used. sEVs were characterised using nanoparticle tracking analysis technology and western blot. CD20 content was also analysed by enzyme-linked immunoassay, and complement-dependent cytotoxicity of rituximab was investigated. 7,8-Dihydroxyflavone (7,8-DHF) was used as a TrkB agonist. In vivo role of sEVs was evaluated in a xenograft model. RESULTS: sEVs production varied significantly between DLBCL cells, independently of subtype. CD20 level was consistent with that of parental cells. Higher CD20 expression was found in sEVs after TrkB activation, with a trend in increasing their concentration. sEVs determined in vitro and in vivo protection from rituximab, which seemed CD20 level-dependent; the protection was enhanced when sEVs were produced by 7,8-DHF-treated cells. CONCLUSIONS: DLBCL-derived sEVs have the differential capacity to interfere with immunotherapy, which could be enhanced by growth factors like neurotrophins. Evaluating the sEV CD20 level could be useful for disease monitoring.

Continuous MYD88 Activation Is Associated With Expansion and Then Transformation of IgM Differentiating Plasma Cells.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

A reduced panel of eight genes (ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53) as an estimator of the tumor mutational burden in chronic lymphocytic leukemia.

INTRODUCTION: Mutational complexity or tumor mutational burden (TMB) influences the course of chronic lymphocytic leukemia (CLL). However, this information is not routinely used because TMB is usually obtained from whole genome or exome, or from large gene panel high-throughput sequencing. METHODS: Here, we used the C-Harrel concordance index to determine the minimum panel of genes for which mutations predict treatment-free survival (TFS) as well as large resequencing panels. RESULTS: An eight gene estimator was defined encompassing ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53. TMB estimated from either a large panel of genes or the eight gene estimator was increased in treated patients or in those with a short TFS (<2 years), unmutated IGHV gene or with an unfavorable karyotype. Being an independent prognostic parameter, any mutation in the eight gene estimator predicted a shorter TFS better than Binet stage and IGHV mutational status among patients with an apparently non-progressive disease (TFS >6 months). Strikingly, the eight gene estimator was also highly informative for patients with Binet stage A CLL or with a good prognosis karyotype. CONCLUSION: These results suggest that the eight gene estimator, that is easily achievable by high-throughput resequencing, brings robust and valuable information that predicts evolution of untreated patients at diagnosis better than any other parameter.

BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells.

Evading apoptosis and sustained survival signaling pathways are two central hallmarks of B-cell chronic lymphocytic leukemia (B-CLL) cells. In this regard, nurse-like cells (NLC), the monocyte-derived type 2 macrophages, deliver stimulatory signals via B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL), and the C-X-C Motif Chemokine Ligand 12 (CXCL12). Previously, we demonstrated that brain-derived neurotrophic factor (BDNF) protects B-CLL cells from spontaneous apoptosis by activating the oncogenic complex NTSR2-TrkB (neurotensin receptor 2-tropomyosin-related kinase receptor B), only overexpressed in B-CLL cells, inducing anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression and Src kinase survival signaling pathways. Herein, we demonstrate that BDNF belongs to the NLC secretome and promotes B-CLL survival. This was demonstrated in primary B-CLL co-cultured with their autologous NLC, compared to B-CLL cells cultured alone. Inhibition of BDNF in co-cultures, enhances B-CLL apoptosis, whereas its exogenous recombinant activates pro-survival pathways in B-CLL cultured alone (i.e. Src activation and Bcl-2 expression), at a higher level than those obtained by the exogenous recombinant cytokines BAFF, APRIL and CXCL12, the known pro-survival cytokines secreted by NLC. Together, these results showed that BDNF release from NLC trigger B-CLL survival. Blocking BDNF would support research strategies against pro-survival cytokines to limit sustained B-CLL cell survival.

Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.

Adult Bone Marrow Three-Dimensional Phenotypic Landscape of B-Cell Differentiation.

BACKGROUND: The different B-cell subsets in human bone marrow result from a dynamic equilibrium between endogenous production, B-cell bone marrow reentry and terminal plasma cell differentiation. Our aim was to define and quantify the different medullary B-cell subsets. METHODS: A series of 32 normal adult bone marrows plus 15 normal adult blood samples was studied by nine color flow cytometry (CD10, CD19, CD24, CD27, CD34, CD38, CD45, IgM, and IgD). With the Kaluza software radar plots, two 2D triple parametric histograms (CD10/CD34/CD45 and CD27/IgM/IgD) were set-up to identify six progenitor and five mature B-cell subsets. RESULTS: Very early B-cell progenitors were CD19neg/CD10pos/CD34pos. B-cell progenitors were split into five subsets on the CD10/CD34/CD45 triple parametric histogram, sequentially ordered according to the loss of CD34 and CD10 and acquisition of surface IgM and IgD. CD19pos/CD38low mature B-cells were divided into four subsets on the CD27/IgM/IgD triple parametric histogram, with two stages of naïve B-cells and two CD27hi marginal zone and switched memory B-cell compartments. CD19pos/CD34neg/CD10low immature B-cells were the main bone marrow B-cell subset, accounting for one third of bone marrow B-cells. Transitional B-cells were the only immature bone marrow stage found in the blood. Compared to blood, the bone marrow was enriched in both marginal zone and switched B-cells. CONCLUSION: We provide the first analysis of 3D B-cell differentiation by multicolor flow cytometry leading to propose reference values for each bone marrow and blood B-cell compartment. This warrants further exploration of normal and pathological human B-cell maturation. © 2018 International Clinical Cytometry Society.

Pathology of Nerve Biopsy and Diagnostic Yield of PCR-Based Clonality Testing in Neurolymphomatosis.

Infiltration of the peripheral nervous system (PNS) by lymphoma, called neurolymphomatosis, is a rare condition among the spectrum of lymphoma-associated neuropathies; its diagnosis is challenging. Cerebrospinal fluid (CSF) analysis is of great value, but nerve biopsy (NB) may be necessary to prove invasion by malignant cells. Clonality polymerase chain reaction (PCR)-based analysis is a validated method in the diagnosis of hematological malignancies, but there are very little data on its diagnostic yield on NB samples. We explored the contribution of NB with clonality analysis to the diagnosis of neurolymphomatosis in 15 patients with negative CSF analysis. Moreover, we assessed the performance of clonality testing in a case-control manner, using patients with inflammatory infiltrates on NB as controls. Neurolymphomatosis was the first manifestation of lymphoma in 60% and could be diagnosed on routine histology alone in 40%. Clonality testing showed monoclonal rearrangement in 86.7% and was unsuccessful in 8.1%. Performance of clonality testing was as follows: 92.9% positive predictive value, 90% negative predictive value, 86.7% sensitivity, 94.7% specificity. This study confirms the diagnostic challenge of neurolymphomatosis, the usefulness of NB in patients with negative CSF analysis, and highlights the high yield of PCR-based clonality testing to assess the malignant nature of PNS lymphoid infiltrates.

Normal serum protein electrophoresis and mutated IGHV genes detect very slowly evolving chronic lymphocytic leukemia patients.

More than 35 years after the Binet classification, there is still a need for simple prognostic markers in chronic lymphocytic leukemia (CLL). Here, we studied the treatment-free survival (TFS) impact of normal serum protein electrophoresis (SPE) at diagnosis. One hundred twelve patients with CLL were analyzed. The main prognostic factors (Binet stage; lymphocytosis; IGHV mutation status; TP53, SF3B1, NOTCH1, and BIRC3 mutations; and cytogenetic abnormalities) were studied. The frequencies of IGHV mutation status, cytogenetic abnormalities, and TP53, SF3B1, NOTCH1, and BIRC3 mutations were not significantly different between normal and abnormal SPE. Normal SPE was associated with Binet stage A, nonprogressive disease for 6 months, lymphocytosis below 30 G/L, and the absence of the IGHV3-21 gene rearrangement which is associated with poor prognosis. The TFS of patients with normal SPE was significantly longer than that of patients with abnormal SPE (log-rank test: P = 0.0015, with 51% untreated patients at 5.6 years and a perfect plateau afterward vs. a median TFS at 2.64 years for abnormal SPE with no plateau). Multivariate analysis using two different Cox models and bootstrapping showed that normal SPE was an independent good prognostic marker for either Binet stage, lymphocytosis, or IGHV mutation status. TFS was further increased when both normal SPE and mutated IGHV were present (log-rank test: P = 0.008, median not reached, plateau at 5.6 years and 66% untreated patients). A comparison with other prognostic markers suggested that normal SPE could reflect slowly advancing CLL disease. Altogether, our results show that a combination of normal SPE and mutated IGHV genes defines a subgroup of patients with CLL who evolve very slowly and who might never need treatment.

Impact of cytogenetic abnormalities in adults with Ph-negative B-cell precursor acute lymphoblastic leukemia.

Multiple cytogenetic subgroups have been described in adult Philadelphia chromosome (Ph)-negative B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), often comprising small numbers of patients. In this study, we aimed to reassess the prognostic value of cytogenetic abnormalities in a large series of 617 adult patients with Ph-negative BCP-ALL (median age, 38 years), treated in the intensified Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 trials. Combined data from karyotype, DNA index, fluorescence in situ hybridization, and polymerase chain reaction screening for relevant abnormalities were centrally reviewed and were informative in 542 cases (88%), allowing classification in 10 exclusive primary cytogenetic subgroups and in secondary subgroups, including complex and monosomal karyotypes. Prognostic analyses focused on cumulative incidence of failure (including primary refractoriness and relapse), event-free survival, and overall survival. Only 2 subgroups, namely t(4;11)/KMT2A-AFF1 and 14q32/IGH translocations, displayed a significantly worse outcome in this context, still observed after adjustment for age and after censoring patients who received allogeneic stem cell transplantation (SCT) in first remission at SCT time. A worse outcome was also observed in patients with low hypodiploidy/near triploidy, but this was likely related to their higher age and worse tolerance to therapy. The other cytogenetic abnormalities, including complex and monosomal karyotypes, had no prognostic value in these intensive protocols designed for adult patients up to the age of 60 years.

Molecular analysis of immunoglobulin variable genes supports a germinal center experienced normal counterpart in primary cutaneous diffuse large B-cell lymphoma, leg-type.

BACKGROUND: Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-). OBJECTIVES: As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. METHODS: Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. RESULTS: A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch µ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. CONCLUSION: These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell.

c-Myc dysregulation is a co-transforming event for nuclear factor-κB activated B cells.

While c-Myc dysregulation is constantly associated with highly proliferating B-cell tumors, nuclear factor (NF)-κB addiction is found in indolent lymphomas as well as diffuse large B-cell lymphomas, either with an activated B-cell like phenotype or associated with the Epstein-Barr virus. We raised the question of the effect of c-Myc in B cells with NF-κB activated by three different inducers: Epstein-Barr virus-latency III program, TLR9 and CD40. Induction of c-Myc overexpression increased proliferation of Epstein-Barr virus-latency III immortalized B cells, an effect that was dependent on NF-κB. Results from transcriptomic signatures and functional studies showed that c-Myc overexpression increased Epstein-Barr virus-latency III-driven proliferation depending on NF-κB. In vitro, induction of c-Myc increased proliferation of B cells with TLR9-dependant activation of MyD88, with decreased apoptosis. In the transgenic λc-Myc mouse model with c-Myc overexpression in B cells, in vivo activation of MyD88 by TLR9 induced splenomegaly related to an increased synthesis phase (S-phase) entry of B cells. Transgenic mice with both continuous CD40 signaling in B cells and the λc-Myc transgene developed very aggressive lymphomas with characteristics of activated diffuse large B-cell lymphomas. The main characteristic gene expression profile signatures of these tumors were those of proliferation and energetic metabolism. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype, activated B-cell like diffuse large B-cell lymphomas. This would explain why NF-κB is associated with both indolent and aggressive lymphomas, and opens new perspectives on the possibility of combinatory therapies targeting both the c-Myc proliferating program and NF-κB activation pathways in diffuse large B-cell lymphomas.

Recommendations for accreditation of laboratories in molecular biology of hematologic malignancies.

Over recent years, the development of molecular biology techniques has improved the hematological diseases diagnostic and follow-up. Consequently, these techniques are largely used in the biological screening of these diseases; therefore the Hemato-oncology molecular diagnostics laboratories must be actively involved in the accreditation process according the ISO 15189 standard. The French group of molecular biologists (GBMHM) provides requirements for the implementation of quality assurance for the medical molecular laboratories. This guideline states the recommendations for the pre-analytical, analytical (methods validation procedures, quality controls, reagents), and post-analytical conditions. In addition, herein we state a strategy for the internal quality control management. These recommendations will be regularly updated.